In addition to the various ways different Hsp40s and other co-chaperones influence prions, highly homologous Hsp70 proteins also affect different prions differently. Constitutively expressed cytosolic Ssa1p and Ssa2p are 98% identical, and stress-inducible Ssa3p and Ssa4p are 88% identical to each other and 80% identical to Ssa1/2p. Remarkably, elevating expression of Ssa1p cures [URE3] but similarly elevating Ssa2p does not, and neither treatment affects [PSI+
Additionally, [URE3] is weaker in cells lacking Ssa2p, but not in cells lacking Ssa1p,83,84
] is weaker in cells lacking Ssa1p but not in cells lacking Ssa2p.20,84
These data show that despite their nearly identical structures Ssa1p and Ssa2p have distinct activities, and suggest that propagation of the different prions relies on different Hsp70 functions.
Unlike the opposing effects Ssa1p and Ssa2p have on [URE3], increasing expression of any of the four Ssa proteins inhibits curing of [PSI+
] by overexpressed Hsp104 and increases frequency of appearance of [PSI+
] when Sup35p prion domain is overexpressed.52,85
These data point to a general [PSI+
]-promoting activity of Ssa protein by antagonizing Hsp104 or enhancing assembly of Sup35p amyloid, although biochemical data suggest Ssa1p antagonizes amyloid formation. As an exception, certain variants of [PSI+
] that form atypically larger aggregates respond oppositely to increases in abundance of Ssa1p and Hsp104. Efficient propagation of these prion variants under non-selective conditions requires increased Hsp104, which was interpreted to mean they are less susceptible to disaggregation by the Hsp104 machinery and thus require increased Hsp104 activity.86,87
Increasing Ssa1p was proposed to adversely affect propagation of these variants by further enhancing aggregation, which leads to increased destabilization. A very recent report shows that prions of a Sup35-GFP fusion protein can be cured by overexpressing Ssa1p.88
What makes the GFP fusion protein prion sensitive to inhibition by Ssa1p, unlike the native Sup35p prion used to induce its formation, was not investigated, but it is possible it also forms larger than normal prion aggregates. In contrast to the Ssa proteins, increasing or depleting Ssb Hsp70 subfamily protein shows Ssb protein consistently as a [PSI+
] prion antagonist.89,90
One observation from these studies is that under different conditions the same Hsp70 can inhibit or promote propagation of the same prion. Since individual chaperones are components of complex machineries, and some even regulate expression of others, care should be used in interpreting data from overexpression studies as being due to simple mass action effects of increasing a particular enzymatic activity. In fact, with regard to Hsp104 and Hsp40 the effects of overexpression are inconsistent with a direct effect of the elevated chaperone activities on prion aggregates. Opposing effects could reflect differences in how a single elevated protein alters different machineries that influence processes of nucleation, assembly and fragmentation of prion polymers. As chaperones are involved in many cellular processes, the effects on prions could be through alteration of cellular processes, possibly connected to the normal functions or interacting partners of the prion protein, that specifically affect propagation of certain prions.
We developed a more simplified system to assess function of any individual Hsp70 by constructing yeast strains lacking all the Ssa proteins. We found that certain Hsp70s from primates and plants support both cell growth and prion propagation, and that they varied widely in these abilities.84,91
In line with phenotypes of cells lacking individual Ssa proteins, [PSI+
] is normal in cells expressing only Ssa1p and somewhat weaker in cells expressing only Ssa2p. Although Ssa3p does not support growth as well as Ssa1/2p, [PSI+
] is stronger in cells expressing only Ssa3p. [PSI+
] is considerably weak but stable in cells expressing Ssa4p, which is weakest at supporting cell growth. In contrast, [URE3] was strong but mitotically unstable in Ssa1p-expressing cells, normal in cells expressing Ssa2p, weak and unstable in cells expressing Ssa3p, and weak but more stable in Ssa4p-expressing cells. Thus, for these Ssa Hsp70s there is no correlation between ability to support growth and prion propagation, and all but Ssa4p have opposite effects on the two prions. The former observation could mean that the different Ssa proteins have different abilities to perform in essential processes that don't affect prion propagation. The latter reveals subtle functional differences among the Hsp70s that might be due to differences in specificity of interactions with substrates or various co-chaperones. These data also show that despite the similar [PSI+
] promoting effect of elevating abundance of any of these four Hsp70s, when expressed at physiological levels each has unique activities that influence prions in both positive and negative ways.
Prion “strength” and mitotic stability are related to the ratio of soluble to insoluble prion protein and the number of prions per cell, respectively.92,93
The solubility is determined by rate of polymer growth and depends on the speed that soluble protein adds to prion polymers and the number of ends that recruit it. The number of prions per cell, which determines the number of ends, reflects the rate of replication due to fragmentation of prion polymers. Therefore, noticeable changes in strength and stability phenotypes provide clues to whether growth or replication processes are affected.93
Although factors that affect prions can influence both growth and replication, these processes can be affected separately. For example, the unstable but strong [URE3] phenotype in cells expressing only Ssa1p probably reflects reduced prion replication, which leads to fewer prions per cell and an increased chance that daughter cells fail to inherit prions, yet the strong phenotype likely indicates normal or even accelerated polymer growth despite the reduced number of polymer ends. Conversely, the weak but stable phenotype of [URE
] cells expressing only Ssa4p could reflect reduced growth of prion polymers but normal replication that maintains a high number of prions per cell. Here the implication is that Ssa1p inhibits or functions less efficiently at processes involving Ure2p polymer fragmentation, while Ssa4p inhibits addition of Ure2p monomers to polymers or is less efficient at promoting polymer growth.
Chaperone alterations that lead to inhibition of fragmentation, and therefore prion replication, likely occur through an effect on the Hsp104 machinery, which could be direct or by limiting interaction of Hsp104 with prions as substrates. In prion aggregates purified from [PSI+
] cells, a molar ratio of Hsp70 (Ssa1/2p) to Sup35p of 1:2 indicates Hsp70 binds along entire Sup35p polymers and might help stabilize them.53
Hsp70s also could act to promote or inhibit growth of prions by binding at the ends of polymers.
The specificity of Hsp70 action on the different prions might be direct and determined by differences in affinity of Hsp70s for the different prion proteins as substrates, although the homology of Ssa1/2p substrate binding domains argues against it. Alternatively, as mentioned, many observations suggest Hsp70 effects on prions are due more to differences in the way they interact with other components of the chaperone machinery than to differences in intrinsic Hsp70 activity. Hsp40s also vary in substrate specificity and they interact differently with different Hsp70s and in their capacity to influence Hsp70 activity. For example, the differences in ways Sis1p and Ydj1p influence [PIN+
] could be because they bind different regions of Rnq1p or because they differ in ability to regulate Ssa protein activity.73,78,81
Thus, while both Hsp70 and Hsp40 recognize prions as substrates, it is likely that specific Hsp70/40 combinations determine specificity for effects on different prions. These combinations are further amplified as different NEFs also affect the system. The curing effect of the NEFs Fes1p and Sse1p on [PSI+
] and [URE3], respectively, appear to be mediated through Hsp70,50,67
as does a requirement of Sse1p for induction of [PSI+
] when Sup35p is overexpressed.94
TPR co-chaperones also could affect prions through interactions with Hsp90, which has been shown to reduce induction of [PSI+
] by Sup35p overexpression.94
In considering the distinctions in chaperone-prion interactions, it has been suggested that prions can be “typed” on the basis of the ways the different chaperones affect their propagation.67
However, the large number of combinations makes categorizing all the effects tricky, and since each prion has certain unique responses to chaperones, correlations are difficult to define (see ).
Effects of altered chaperones on prion phenotypes
Finally, although all amyloid-forming yeast prions tested assemble into in-register parallel beta-sheet conformations,95–99
it should be pointed out that differences in structural organization of polymers formed by different prion proteins, as well as different strains of the same prion, could influence their susceptibility to being recognized or acted on by chaperones. For example Ure2p might form more rigid structures than Sup35p, and thus rely more heavily on particular combinations of chaperone components that provide strongest fragmentation activity.
Defining the precise molecular mechanisms underlying prion propagation is challenging because altering chaperones affects prions in very many ways and the complex and often unpredictable ways chaperones interact with each other and with substrates in the cell make it difficult to simplify. Moreover, chaperones have roles in many diverse cellular processes so they can be expected to affect prions in indirect ways. Although the complexity is daunting, the strict requirement of Hsp104 and it's well-defined role in prion replication provides a basis for inferring that most chaperone alterations that influence the prion replication process are likely to be mediated through an effect on the Hsp104 machinery. At least for [PSI+], solubilization activity of Hsp104, which depends on Hsp70 and Hsp40, is critical for prion propagation, implying that Hsp104 does not act alone in [PSI+] replication. There is only one Hsp104 but many chaperones that influence the Hsp104 machinery. As a central player and regulatory target, Hsp70 links these chaperones to this machine.